The present invention relates to an arrangement for two-dimensional separation of a complex mixture of e.g., peptides, and methods for producing such arrangement by liquid chromatography. In particular, the invention relates to reversed phase liquid chromatography (RPLC) followed by capillary electrophoresis (CE) spraying directly into a mass spectrometer.
Two-dimensional liquid chromatographic/mass-spectrometric methods (LC/MS) start playing a key role in proteomics research and applications due to the fact that 2D LC has been found to overcome some of the limitations crucial to 2D Polyacrylamid Gel Electrophoresis (PAGE). The main advantages of this process are automation, shorter analysis time, higher sensitivity and increased reproducibility. Dilute samples can be concentrated on-column and coupling with different detection systems is possible, like UV, LIF and especially MS leading to quicker identification and quantitation.
Analysis at the peptide level is preferable for several reasons but mainly because peptides are initially more soluble in a wider variety of solvents and are easier to separate than the parent proteins. There is however a disadvantage in working with peptides, which is the increase in the number of species that have to be solved, thus demanding higher resolution during separation and making the use of available pre-fractionation techniques more important.
The basic idea of an on-line 2D-LC system is to have a slow separation in the first dimension and a fast separation in the second dimension. In order to match the requirements for high resolution in the second dimension without reducing the sampling rate or slowing down the first dimension, at least two second dimension columns need to be eluted in parallel. The most reported but also the most applied and commercialized 2D-LC approach is ion exchange chromatography (IEX) followed by RPLC. The two techniques are fully orthogonal, the first based on a charge separation mechanism (salt elution steps), the second based on hydrophobicity (gradient elution with organic solvent). IEX is not compatible with MS, thus it is employed as first dimension. Fractions from IEX are trapped and washed on one or two parallel enrichment columns, then subjected to second separation by RPLC (one or two parallel columns), which can be coupled directly to MS typically trough ESI interface. A limitation of this approach is still the overall long time of analysis.
Theoretically, one of the combinations offering most advantages is LC followed by Capillary Zone Electrophoresis (CZE) but thus far, nothing has been commercialized for this specific coupling and little can be retrieved in the literature. These two techniques are also orthogonal and compatible between them and with mass spectrometry. They both provide high resolution increasing the total peak capacity and together can be faster than any other 2D combination.
The rate at which CZE separations can be carried out allows the continuous sampling of the effluent from the first column into the second, completing the 2D analysis in the time it takes to complete the first dimension, that is the time it takes to perform an RPLC separation. The critical issue that has historically slowed the development of this strategy is probably the interface between the two separation techniques. Interesting ideas, not always intended for this specific coupling, are more or less valid solutions. A few alternative methods are described in the prior art, like that based on a mechanical valve and those based on a flow gating or optical gating concept. Another concept could also be employed based on dispensing, by one of several means, droplets of defined volumes from a continuous flow into for example a capillary for CE. Other concepts, e.g., based on control of EOF (Electro Osmotic Flow) are described in the prior art. But in all cases, besides individual limitations, a common disadvantage still remains, that is most of the effluent is sent to waste with risk to lose information and sensitivity is certainly not improved.
On-column preconcentration in capillary electrophoresis is known in the art, however, these known methods require either discontinuous buffer systems, e.g., for isotachophoresis and field amplification, or the use of adsorbing or binding materials generally also requiring a change in the mobile phase or buffer for desorption. Electrokinetic trapping has also been reported in the prior art, where proteins are trapped on silica particles under an applied electric field and eluted by pressure in absence of electric field. A fluidic electrocapture device based on the equilibrium between hydrodynamic and electrical forces has also been described. However, this was meant for one-step sample cleanup of peptides and proteins before e.g., Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry (MALDI-MS) or preconcentration before one-dimensional off-line analysis by capillary electrophoresis, and in fact, it only helps to prove the principle on which the interface of this invention is based